Aleutian disease: Risk factors and ImmunAD strategy for genetic improvement of tolerance in American mink (Neogale vison).

Aleutian disease (AD) is a devastating infectious disease in American mink (Neogale vison) industry caused by Aleutian mink disease virus (AMDV). Two crucial steps toward controlling infectious diseases in farm animals are: (i) assessment of the infection risk factors to minimize the likelihood of infection and (ii) selection of animals with superior immune responses against pathogens to build tolerant farms. This study aimed to investigate AD risk factors and evaluate a novel "ImmunAD" approach for genetic improvement of AD tolerance. Phenotypic records and pedigree information of 1,366 and 24,633 animals were included in this study. The risk of animal's age, sex, color type, and year of sampling on AMDV infection was assessed using a logistic regression model and counter immune-electrophoresis (CIEP) test results. ImmunAD phenotype was calculated based on AMDVG enzyme-linked immunosorbent assay (ELISA) and CIEP test results, and breeding values for ImmunAD were estimated using an animal model. Animals were classified into high-coordinated (HCIR), average-coordinated (ACIR), and low-coordinated immune responders (LCIR) using ImmunAD's breeding values, and the impact of selection of HCIR on live grade of pelt quality (PQ), harvest weight (HW), and harvest length (HL) breeding values were evaluated. Age of > 1 year, male sex, and year of sampling were identified as significant risk factors of AD (p < 0.05). A moderate-to-high heritability (0.55±0.07) was estimated for ImmunAD, while a higher heritability was observed among the CIEP-positive animals (0.76±0.06). Significantly higher breeding values were observed for PQ and HL among HCIR than those for LCIR and ACIR (p < 0.05). Our findings indicate the critical role of male breeders in AD distribution within mink farms. Regular screening of AD in male breeders before pairing them with females during breeding seasons can help disease control. ImmunAD strategy can be applied to genetic improvement of AD tolerance, with favorable impacts on some growth and production traits. Higher genetic gains can be achieved in populations with higher AD seroprevalences.

Diverse amdoparvoviruses infection of farmed Asian badgers (Meles meles).

Amdoparvoviruses infect various carnivores, including mustelids, canids, skunks, and felids. Aleutian mink disease virus (AMDV) belongs to the prototypical species Amdoparvovirus carnivoran1. Here, we identified a novel amdoparvovirus in farmed Asian badgers (Meles meles), and we named this virus "Meles meles amdoparvovirus" (MMADV). A total of 146 clinical samples were collected from 134 individual badgers, and 30.6% (41/134) of the sampled badgers tested positive for amdoparvovirus by PCR. Viral DNA was detected in feces, blood, spleen, liver, lung, and adipose tissue from these animals. Viral sequences from eight samples were determined, five of which represented nearly full-length genome sequences (4,237-4,265 nt). Six serum samples tested positive by PCR, CIEP, and IAT, four of which had high antibody titers (> 512) against AMDV-G. Twenty-six of the 41 amdoparvovirus-positive badgers showed signs of illness, and necropsy revealed lesions in their organs. Sequence comparisons and phylogenetic analysis of the viral NS1 and VP2 genes of these badger amdoparvoviruses showed that their NS1 proteins shared 62.6%-88.8% sequence identity with known amdoparvoviruses, and they clustered phylogenetically into two related clades. The VP2 proteins shared 76.6%-97.2% identity and clustered into two clades, one of which included raccoon dog and arctic fox amdoparvovirus (RFAV), and the other of which did not include other known amdoparvoviruses. According to the NS1-protein-based criterion for parvovirus species demarcation, the MMADV isolate from farm YS should be classified as a member of a new species of the genus Amdoparvovirus. In summary, we have discovered a novel MMADV and other badger amdoparvoviruses that naturally infect Asian badgers and are possibly pathogenic in badgers.

Strong selection signatures for Aleutian disease tolerance acting on novel candidate genes linked to immune and cellular responses in American mink (Neogale vison).

Aleutian disease (AD) is a multi-systemic infectious disease in American mink (Neogale vison) caused by Aleutian mink disease virus (AMDV). This study aimed to identify candidate regions and genes underlying selection for response against AMDV using whole-genome sequence (WGS) data. Three case-control selection signatures studies were conducted between animals (N = 85) producing high versus low antibody levels against AMDV, grouped by counter immunoelectrophoresis (CIEP) test and two enzyme-linked immunosorbent assays (ELISA). Within each study, selection signals were detected using fixation index (FST) and nucleotide diversity (θπ ratios), and validated by cross-population extended haplotype homozygosity (XP-EHH) test. Within- and between-studies overlapping results were then evaluated. Within-studies overlapping results indicated novel candidate genes related to immune and cellular responses (e.g., TAP2, RAB32), respiratory system function (e.g., SPEF2, R3HCC1L), and reproduction system function (e.g., HSF2, CFAP206) in other species. Between-studies overlapping results identified three large segments under strong selection pressure, including two on chromosome 1 (chr1:88,770-98,281 kb and chr1:114,133-120,473) and one on chromosome 6 (chr6:37,953-44,279 kb). Within regions with strong signals, we found novel candidate genes involved in immune and cellular responses (e.g., homologous MHC class II genes, ITPR3, VPS52) in other species. Our study brings new insights into candidate regions and genes controlling AD response.

Asymptomatic viral infection is associated with lower host reproductive output in wild mink populations.

Many endemic viruses circulate in populations without hosts showing visible signs of disease, while still having the potential to alter host survival or reproduction. Aleutian Mink Disease Virus (AMDV) circulates in many American mink (Neogale vison) populations in its native and introduced ranges. In this study, we analysed how AMDV infection in female American mink affects the reproduction of a feral population. Females infected with AMDV delivered significantly smaller litters (5.8 pups) than uninfected females (6.3 pups), meaning their litter size was reduced by 8%. Larger females and yearling females had larger litters than smaller and older females. There were no significant differences in whole litter survival between infected and uninfected females; however, offspring survival until September or October within litters of infected females was 14% lower than that within those of uninfected females. This negative link between infection and reproductive output means that Aleutian disease could seriously affect the wild mink population. This study increases our understanding of the threats posed by the spread of viruses to wildlife from farm animals or humans, highlighting that viruses circulating in wildlife, even in the absence of clinical manifestation, can be important drivers of population dynamics in wildlife.

First Report of Skunk Amdoparvovirus (Species Carnivore amdoparvovirus 4) in Europe in a Captive Striped Skunk (Mephitis mephitis).

Skunk amdoparvovirus (Carnivore amdoparvovirus 4, SKAV) is closely related to Aleutian mink disease virus (AMDV) and circulates primarily in striped skunks (Mephitis mephitis) in North America. SKAV poses a threat to mustelid species due to reported isolated infections of captive American mink (Neovison vison) in British Columbia, Canada. We detected SKAV in a captive striped skunk in a German zoo by metagenomic sequencing. The pathological findings are dominated by lymphoplasmacellular inflammation and reveal similarities to its relative Carnivore amdoparvovirus 1, the causative agent of Aleutian mink disease. Phylogenetic analysis of the whole genome demonstrated 94.80% nucleotide sequence identity to a sequence from Ontario, Canada. This study is the first case description of a SKAV infection outside of North America.

Genetic and phenotypic correlations between Aleutian disease tests with body weight, growth, and feed efficiency traits in mink.

The ineffectiveness of vaccination, medicine, and culling strategy leads mink farmers to control Aleutian disease (AD) by selecting AD-resilient mink based on AD tests. However, the genetic background of AD tests and their correlations with economically important or AD-resilient traits are limited. This study estimated the genetic and phenotypic correlations between four AD tests and seven body weight (BW) traits, six growth parameters from the Richards growth model, and eight feed-related traits. Univariate models were used to test the significance (P < 0.05) of fixed effects (sex, color type, AD test year, birth year, and row-by-year), random effects (additive genetic, maternal genetic, and permanent environmental), and a covariate of age using ASReml 4.1. Likewise, pairwise bivariate analyses were conducted to estimate the phenotypic and genetic correlations among the studied traits. Both antigen- and virus capsid protein-based enzyme-linked immunosorbent assay tests (ELISA-G and ELISA-P) showed significant (P < 0.05) moderate positive genetic correlations (±SE) with maturation rate (from 0.36 ± 0.18 to 0.38 ± 0.19). ELISA-G showed a significant negative genetic correlation (±SE) with average daily gain (ADG, -0.37 ± 0.16). ELISA-P showed a significant positive moderate genetic correlation (±SE) with off-feed days (DOF, 0.42 ± 0.17). These findings indicated that selection for low ELISA scores would reduce the maturation rate, increase ADG (by ELISA-G), and minimize DOF (by ELISA-P). The iodine agglutination test (IAT) showed significant genetic correlations with DOF (0.73 ± 0.16), BW at 16 weeks of age (BW16, 0.45 ± 0.23), and BW at harvest (HW, -0.47 ± 0.20), indicating that selection for lower IAT scores would lead to lower DOF and BW16, and higher HW. These estimated genetic correlations suggested that the selection of AD tests would not cause adverse effects on the growth, feed efficiency, and feed intake of mink. The estimates from this study might strengthen the previous finding that ELISA-G could be applied as a reliable and practical indicator trait in the genetic selection of AD-resilient mink in AD-positive farms.

The selection of Aleutian disease-resistant individuals based on Aleutian disease (AD) tests is seen as a potential method to control AD effectively. However, the knowledge regarding the genetic background of AD tests is limited. This study estimated the genetic and phenotypic correlations between Aleutian disease tests and body weight, growth, and feed-related traits in mink. The estimates in this study indicated that the growth, feed efficiency, and feed intake of mink would not be adversely influenced by the selection of AD tests. In the meantime, the estimates further illustrate that the antigen-based enzyme-linked immunosorbent assay test could be applied as the most reliable and practical indicator trait to select AD-resilient mink in AD-positive farms.

Long-term antibody production and viremia in American mink (Neovison vison) challenged with Aleutian mink disease virus.

BACKGROUND: Selecting American mink (Neovison vison) for tolerance to Aleutian mink disease virus (AMDV) has gained popularity in recent years, but data on the outcomes of this activity are scant. The objectives of this study were to determine the long-term changes in viremia, seroconversion and survival in infected mink. Mink were inoculated intranasally with a local isolate of Aleutian mink disease virus (AMDV) over 4 years (n = 1742). The animals had been selected for tolerance to AMDV for more than 20 years (TG100) or were from herds free of AMDV (TG0). The progenies of TG100 and TG0, and their crosses with 25, 50 and 75% tolerance ancestry were also used. Blood samples were collected from each mink up to 14 times until 1211 days post-inoculation (dpi) and were tested for viremia by PCR and for anti-AMDV antibodies by counter-immunoelectrophoresis (CIEP). Viremia and CIEP status were not considered when selecting replacements. Low-performing animals were pelted and the presence of antibodies in their blood and antibody titer were measured by CIEP, and viremia and viral DNA in seven organs (n = 936) were tested by PCR. RESULTS: The peak incidences of viremia (66.7%) and seropositivity (93.5%) were at 35 dpi. The incidence of viremia decreased over time while the incidence of seroconversion increased. The least-squares means of the incidence of PCR positive of lymph node (0.743) and spleen (0.656) were significantly greater than those of bone marrow, liver, kidneys, lungs and small intestine (0.194 to 0.342). Differences in tolerant ancestry were significant for every trait measured. Incidences of viremia over time, terminal viremia, seropositivity over time, AMDV DNA in organs and antibody titer were highest in the susceptible groups (TG0 or TG25) and lowest in the tolerant groups (TG100 or TG75). CONCLUSION: Previous history of selection for tolerance resulted in mink with reduced viral replication and antibody titer. Viremia had a negative effect and antibody production had a positive effect on survival and productivity.

Mechanisms behind the varying severity of Aleutian mink disease virus: Comparison of three farms with a different disease status.

Aleutian mink disease virus (AMDV) is distributed widely among mink farms and wild mustelids despite ongoing attempts to stop the spread. The severity of Aleutian disease (AD) varies from subclinical to fatal but the reasons for its varying severity are complex and unclear. Recently, breeding of tolerant mink has drawn attention as the possible solution to reduce the effects of AD in farms. The aim of this study was to gather information on the effects of breeding based on overall health, production traits, and antibody titer on AD severity by comparing a positive farm (farm 1) that has been breeding for tolerance in mink to an infected farm without tolerance selection, and an AMDV-free farm. During the 2.5-year follow-up, the mink in farm 1 remained mostly free of clinical AD, had normal pelt quality and litter size, and had low virus copy numbers in tissues and low antibody titers in ELISA. In histopathological studies, most of the farm 1 mink had no/mild lesions in their kidneys. 29-43% of the mink were ELISA negative but PCR positive throughout the follow-up and frequent changes in virus strains and coinfections were observed. Several differences in gene expression between animals from different farms were also detected. These results indicate that the disease burden of AMDV can be reduced, with seemingly normal health and production rates, despite continual circulation of ADMV in cases where eradication attempts are unsuccessful.

AMDV Vaccine: Challenges and Perspectives.

Aleutian mink disease virus (AMDV) is known to cause the most significant disease in the mink industry. It is globally widespread and manifested as a deadly plasmacytosis and hyperglobulinemia. So far, measures to control the viral spread have been limited to manual serological testing for AMDV-positive mink. Further, due to the persistent nature of this virus, attempts to eradicate Aleutian disease (AD) have largely failed. Therefore, effective strategies to control the viral spread are of crucial importance for wildlife protection. One potentially key tool in the fight against this disease is by the immunization of mink against AMDV. Throughout many years, several researchers have tried to develop AMDV vaccines and demonstrated varying degrees of protection in mink by those vaccines. Despite these attempts, there are currently no vaccines available against AMDV, allowing the continuation of the spread of Aleutian disease. Herein, we summarize previous AMDV immunization attempts in mink as well as other preventative measures with the purpose to shed light on future studies designing such a potentially crucial preventative tool against Aleutian disease.

Genetic and phenotypic parameters for Aleutian disease tests and their correlations with pelt quality, reproductive performance, packed-cell volume, and harvest length in mink.

Aleutian disease (AD), caused by the Aleutian mink disease virus (AMDV), is a major health concern that results in global economic losses to the mink industry. The unsatisfactory outcome of the culling strategy, immunoprophylaxis, and medical treatment in controlling AD have urged mink farmers to select AD resilient mink based on several detection tests, including enzyme-linked immunosorbent assay (ELISA), counterimmunoelectrophoresis (CIEP), and iodine agglutination test (IAT). However, the genetic analysis of these AD tests and their correlations with pelt quality, reproductive performance, packed-cell volume (PCV), and harvest length (HL) have not been investigated. In this study, data on 5,824 mink were used to estimate the genetic and phenotypic parameters of four AD tests, including two systems of ELISA, CIEP, and IAT, and their genetic and phenotypic correlations with two pelt quality, five female reproductive performance, PCV, and HL traits. Significances (P < 0.05) of fixed effects (sex, year, dam age, and color type), covariates (age at harvest and blood sampling), and random effects (additive genetic, permanent environmental, and maternal effects) were determined under univariate models using ASReml 4.1 software. The genetic and phenotypic parameters for all traits were estimated under bivariate models using ASReml 4.1 software. Estimated heritabilities (±SE) were 0.39 ± 0.06, 0.61 ± 0.07, 0.11 ± 0.07, and 0.26 ± 0.05 for AMDV antigen-based ELISA (ELISA-G), AMDV capsid protein-based ELISA, CIEP, and IAT, respectively. The ELISA-G also showed a moderate repeatability (0.58 ± 0.04) and had significant negative genetic correlations (±SE) with reproductive performance traits (from -0.41 ± 0.16 to -0.49 ± 0.12), PCV (-0.53 ± 0.09), and HL (-0.45 ± 0.16). These results indicated that ELISA-G had the potential to be applied as an indicator trait for genetic selection of AD resilient mink in AD endemic ranches and therefore help mink farmers to reduce the adverse effects caused by AD.

Detection of selection signatures for response to Aleutian mink disease virus infection in American mink.

Aleutian disease (AD) is the most significant health issue for farmed American mink. The objective of this study was to identify the genomic regions subjected to selection for response to infection with Aleutian mink disease virus (AMDV) in American mink using genotyping by sequencing (GBS) data. A total of 225 black mink were inoculated with AMDV and genotyped using a GBS assay based on the sequencing of ApeKI-digested libraries. Five AD-characterized phenotypes were used to assign animals to pairwise groups. Signatures of selection were detected using integrated measurement of fixation index (FST) and nucleotide diversity (θπ), that were validated by haplotype-based (hap-FLK) test. The total of 99 putatively selected regions harbouring 63 genes were detected in different groups. The gene ontology revealed numerous genes related to immune response (e.g. TRAF3IP2, WDR7, SWAP70, CBFB, and GPR65), liver development (e.g. SULF2, SRSF5) and reproduction process (e.g. FBXO5, CatSperß, CATSPER4, and IGF2R). The hapFLK test supported two strongly selected regions that contained five candidate genes related to immune response, virus-host interaction, reproduction and liver regeneration. This study provided the first map of putative selection signals of response to AMDV infection in American mink, bringing new insights into genomic regions controlling the AD phenotypes.

Development of an antigen-capture enzyme-linked immunosorbent assay for diagnosis of Aleutian mink disease virus.

Aleutian mink disease (AMD), caused by Aleutian mink disease virus (AMDV), is a very important infectious disease of mink. Currently, elimination of antibody- or antigen-positive animals is the most successful strategy for eradicating AMD, but the claw-cutting method of blood sampling is difficult to perform and painful for the animal. In this study, we aimed to establish an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) method for the efficient detection of AMDV antigens using fecal samples. A purified mouse monoclonal antibody (mAb) was used as the capture antibody, and a rabbit polyclonal antibody (pAb) was used as the detection antibody. The assay was optimized by adjusting a series of parameters. Using a cutoff value of 0.205, the limit of detection of the AC-ELISA for strain AMDV-G antigen was 2 µg/mL, and there was no cross-reaction with other mink viruses. The intra- and inter-assay standard deviations were below 0.046, and the correlation of variance (CV) values were 1.24-7.12% when testing fecal samples. Compared with conventional PCR results, the specificity and sensitivity were 91.5% and 90.6%, respectively, and the concordance rate between the two methods was 91.1%.

Aleutian mink disease: Spatio-temporal variation of prevalence and influence on the feral American mink.

Pathogens are one of the factors driving wildlife population dynamics. The spread of pathogens in wildlife is currently highly related to the transmission of pathogens from farmed animals, which has increased with the constant development of farming. Here, we analysed the spatio-temporal variation in the prevalence of Aleutian mink disease virus (AMDV) antibodies in feral American mink (Neovison vison) populations in Poland (1,153 individuals from nine sites) in relation to mink farming intensity. AMDV was detected in feral mink at all study sites and the prevalence ranged from 0.461 in the northern region to 0.826 in the western region. Mink males and adults were infected more often than females and subadults; the infection was also more frequent during the mink breeding season than during non-breeding. The prevalence of AMDV changed non-linearly in consecutive years and the peak of prevalence was every 3-4 years. The predicted AMDV prevalence was low at sites where the number of farmed mink was also low and increased linearly with the increase in the number of mink kept on farms. The predicted AMDV prevalence at sites with low mink farming intensity strongly varied between years, whereas at sites with high mink farming intensity, the predicted prevalence did not change significantly. AMDV infection affected the mink's body condition and caused an increase in the size of the spleen, liver and kidneys. This study shows that Aleutian mink disease strongly affects feral mink but the spatio-temporal variation of its prevalence is complex and partly related to the transmission of the virus from farmed mink to feral populations. The study highlights the complexity of AMDV circulation in feral mink populations and implicates a potential spillover of the virus to native species.

Dietary supplementation of Ascophylum nodosum improved kidney function of mink challenged with Aleutian mink disease virus.

BACKGROUND: Feed additives which can ease the negative effects of infection by the Aleutian mink disease virus (AMDV) are of interest to mink farmers. The effects of kelp meal (Ascophylum nodosum) supplementation on immune response, virus replication and blood parameters of mink inoculated with AMDV were assessed. AMDV-free black mink (n = 75) were intranasally inoculated with a local strain of AMDV and fed a commercial pellet supplemented with kelp meal at the rates of 1.5% or 0.75% of the feed or were kept as controls (no kelp) for 451 days. Blood was collected on days 0 (pre-inoculation), 31, 56, 99, 155, 366 and 451 post-inoculation (dpi). RESULTS: No significant difference was observed among the treatments for the proportion of animals positive for antibodies against the virus measured by the counter-immunoelectrophoresis (CIEP), viremia measured by PCR, antibody titer measured by quantitative ELISA, total serum protein measured by a refractometer or elevated levels of gamma globulin measured by iodine agglutination test at the sampling occasions. At the termination of the experiment on 451 dpi, there were no differences among treatments for antibody titer measured by CIEP, total serum protein, albumin, globulins, albumin:globulin ratio, alkaline phosphatase, gamma-glutamyl transferase, and proportions of PCR positive spleen, lymph node or bone marrow samples, but blood urea nitrogen and creatine levels were significantly lower in the 1.5% kelp supplemented group than in the controls. CONCLUSION: Kelp supplementation improved kidney function of mink infected with AMDV with no effect on liver function, immune response to infection by AMDV or virus replication.

Molecular epidemiology of Aleutian mink disease virus causing outbreaks in mink farms from Southwestern Europe: a retrospective study from 2012 to 2019.

BACKGROUND: Aleutian mink disease virus (AMDV) causes major economic losses in fur-bearing animal production. The control of most AMDV outbreaks is complex due to the difficulties of establishing the source of infection based only on the available on-farm epidemiological data. In this sense, phylogenetic analysis of the strains present in a farm may help elucidate the origin of the infection and improve the control and biosecurity measures. OBJECTIVES: This study had the following aims: characterize the AMDV strains from most outbreaks produced at Spanish farms between 2012-2019 at the molecular level, and assess the utility of the combined use of molecular and epidemiological data to track the possible routes of infection. METHODS: Thirty-seven strains from 17 farms were partially sequenced for the NS1 and VP2 genes and analyzed phylogenetically with other strains described worldwide. RESULTS: Spanish AMDV strains are clustered in four major clades that generally show a good geographical correlation, confirming that most had been established in Spain a long time ago. The combined study of phylogenetic results and epidemiological information of each farm suggests that most of the AMDV outbreaks since 2012 had been produced by within-farm reservoirs, while a few of them may have been due to the introduction of the virus through international trade. CONCLUSIONS: The combination of phylogenetic inference, together with epidemiological data, helps assess the possible origin of AMDV infections in mink farms and improving the control and prevention of this disease.

Molecular epidemiology of Aleutian mink disease virus from fecal swab of mink in northeast China.

BACKGROUND: Aleutian mink disease parvovirus (AMDV) causes Aleutian mink disease (AMD), which is a serious infectious disease of mink. The aim of this study was to get a better understanding of the molecular epidemiology of AMDV in northeast China to control and prevent AMD from further spreading. This study for the first time isolated AMDV from fecal swab samples of mink in China. RESULTS: A total of 157/291 (54.0%) of the fecal swab samples were positive for AMDV. Of these, 23 AMDV positive samples were randomly selected for sequence alignment and phylogenetic analysis based on the acquired partial fragments of VP2 gene with the hypervariable region. Comparative DNA sequence analysis of 23 AMDV isolates with a reference nonpathogenic (AMDV-G) strain revealed 8.3% difference in partial VP2 nucleotide sequences. Amino acid alignment indicated the presence of several genetic variants, as well as one single amino acid residue deletion. The most concentrated area of variation was located in the hypervariable region of VP2 protein. According to phylogenetic analysis, the Chinese AMDV strains and the other reference AMDV strains from different countries clustered into three groups (clades A, B and C). Most of the newly sequenced strains were found to form a Chinese-specific group, which solely consisted of Chinese AMDV strains. CONCLUSION: These findings indicated that a high genetic diversity was found in Chinese AMDV strains and the virus distribution were not dependent on geographical origin. Both local and imported AMDV positive species were prevalent in the Chinese mink farming population. The genetic evidence of AMDV variety and epidemic isolates have importance in mink farming practice.

Dose response of black American mink to Aleutian mink disease virus.

INTRODUCTION: Aleutian mink disease virus (AMDV) causes a serious health problem for mink globally. The disease has no cure nor an effective vaccine and selection for tolerance using antibody titer is adopted by many mink farmers. The objective of this study was to investigate the effects of various doses of a local AMDV isolate on the response of black American mink to infection with AMDV. METHODS: Eight black American mink were each inoculated intranasally with 0.5 mL of eight serial 10-fold dilutions (100 to 10-7 ) of a 10% spleen homogenate containing a local AMDV isolate. Blood samples were collected on days 0, 20, 35, 56, 84, 140, and 196 postinoculation (dpi). Anti-AMDV antibodies and viral DNA were tested by counter-immunoelectrophoresis (CIEP) and PCR, respectively. Animals that were PCR or CIEP positive at 196 dpi (n = 41) were killed at 218 dpi, and samples of blood and seven organs were tested by CIEP and PCR. RESULTS: Antibody production persisted in all seroconverted mink until the termination of the experiment, whereas 71.1% of the mink showed short-lived viremia. Significant associations were observed between inoculum dose and the incidence of viremia until 84 dpi which disappeared thereafter, whereas associations between inoculum dose and the incidence of seropositive mink were significant on all sampling occasions. Antibody titer at 218 dpi significantly decreased with decreasing inoculum dose. AMDV DNA was detected in the bone marrow, lymph nodes, and spleen samples of almost all mink inoculated at every dose but was not detected in other organs of some mink. CONCLUSIONS: CIEP is more accurate than PCR for detecting AMDV infection in mink. Using antibody titer in naturally infected mink may not be accurate for the identification of tolerant mink.

Mink Aleutian disease seroprevalence in China during 1981-2017: A systematic review and meta-analysis.

Mink Aleutian disease (AMD) is the first of the three major diseases of fur animals. It is a common immunosuppressive disease in mink farms worldwide, which seriously endangers the development of the mink farming industry. Strengthening the understanding of the positive serum rate and spatial distribution of AMD is of great significance for the prevention and control of disease caused by the Aleutian virus. Therefore, we conducted a systematic review and meta-analysis to assess the seroprevalence of AMD in China. We extracted 45 studies related to the seroprevalence of Chinese AMD, with samples taken between 1981 and 2017. Our systematic review and meta-analysis results show that, during the selected period, the overall positive rate of AMD in China was 55.3% (95% CI 48.5-62.0). The results from subgroups analysis of the potential risk factors showed that the seroprevalence rate of AMD in China in the past 36 years rose from 48% (95% CI 37.0-60.5) in 1981-2009 to 61.4% (95% CI 43.6-79.3) in 2010-2017. The date of the spatial difference in AMD seroprevalence indicated that AMD seroprevalence was unevenly distributed in different regions: the number of mink in eastern China and northeastern China was relatively high, and the seroprevalence rates were 57.9%, (95% CI 46.2-69.7) and 61.3% (95% CI 53.1-69.5), respectively. Central China had the highest seroprevalence rate of AMD at 69.8% (95% CI 64.4-75.2). At the provincial level, the AMD seroprevalence rate in Jiangsu was as high as 96% (95% CI 94.1-97.8), and the AMD seroprevalence rate in Shaanxi was the lowest at 22.1% (95% CI 20.3-23.9). This suggested that the AMD seroprevalence rate in China was unevenly distributed. In other subgroups, the positive rate of AMD in adult mink was higher than in juvenile mink. This implied that the high prevalence of AMD in China was caused by multiple factors. The meta-regression results indicated that the detection method subgroup (P = 0.008) may be the source of heterogeneity. Our data system evaluated the prevalence of Aleutian disease in China in the last 37 years and a preliminary discussion on the risk factors of AMD. It may help prevent and control AMD in China. It is recommended to conduct further epidemiological testing and develop a comprehensive testing plan to determine the risk factors associated with Aleutian disease and improve the Aleutian disease control strategy.

Development of an EvaGreen-based real-time PCR assay for detection of Aleutian mink disease virus.

The objective of this study was to develop a rapid, sensitive and specific EvaGreen (EG)-based real-time PCR assay capable of detecting Aleutian mink disease virus (AMDV) and to evaluate the reliability of the assay for analysis of blood or tissue samples. For this assay, a pair of primers was designed based on a nonstructural protein (NS)-encoding gene of AMDV, and the identity of PCR products was identified based on a melting temperature of 82.8°C. The EG-based real-time PCR assay did not detect canine distemper virus or mink enteritis virus, and the assay could be used to detect Chinese and American AMDV strains, in contrast to a commercial TaqMan kit that could only detect American AMDV strains. The amplification efficiencies of the EG assay were 104.8% for the Chinese strain and 94.4% for the American strain, and the detection limit was 1 copy/µL of AMDV plasmid or 3 pg/µL of viral DNA (Chinese strain). The intra- and inter-assay variation coefficients of melting temperature were all lower than 0.15%, confirming the high reproducibility of the assay. Forty-five clinical blood samples were simultaneously analyzed using the EG real-time PCR, TaqMan kit and conventional PCR, and the detection rates were 91.1%, 0.0% and 86.7%, respectively. Serum samples were also collected from the corresponding blood samples and tested using the counterimmunoelectrophoresis (CIEP) assay, where positive samples accounted for 24.4% of the 45 samples. In conclusion, EG-based real-time PCR is a rapid, sensitive, universal assay that can be effectively utilized as a reliable and specific tool for detection and quantitation of AMDV.

Development and validation of nucleic acid tests to diagnose Aleutian mink disease virus.

Aleutian disease (AD), caused by Aleutian mink disease virus (AMDV), causes significant welfare problems to mink, and financial losses to the farmers. As there is no vaccine or treatment available, reliable diagnostics is important for disease control. Here, we set up a probe-based real-time PCR (NS1-probe-PCR) to detect all strains of AMDV. PCR was validated and compared to two other real-time PCR methods (pan-AMDV- and pan-AMDO-PCR) currently used for AMDV diagnostics in Finland. The NS1-probe-PCR had a similar detection limit of 20 copies/reaction based on plasmid dilution series, and similar or better diagnostic sensitivity, when evaluated using spleen samples from mink, and stool samples from mink and foxes. None of the three PCR tests cross-reacted with other parvoviruses. The NS1-probe-PCR also showed a significantly higher specificity than the pan-AMDO-PCR with spleen samples and the best specificity with stool samples. Furthermore, it produced the results more rapidly than the other two PCRs making it a promising tool for both diagnostic and research purposes.